1. Field of the Invention
The invention relates generally to an assay for detecting an amplification product and more specifically to an assay for detecting the genotype of the amplification product at two or more different allelic sites.
2. Description of Related Art
Nucleic acid sequence analysis is becoming increasingly important in many research, medical, and industrial fields, e.g. Caskey, Science 236: 1223-1228 (1987); Landegren et al, Science, 242: 229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61: 131-156 (1992). The development of several nucleic acid amplification schemes has played a critical role in this trend, e.g. polymerase chain reaction (PCR), Innis et al, editors, PCR Protocols (Academic Press, New York, 1990); McPherson et al, editors, PCR: A Practical Approach (IRL Press, Oxford, 1991); ligation-based amplification techniques, Barany, PCR Methods and Applications 1: 5-16 (1991); and the like.
PCR in particular has become a research tool of major importance with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like, e.g. Arnheim et al (cited above); Gilliland et al, Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan et al, PCR Methods and Applications, 1: 222-228 (1992); Green et al, PCR Methods and Applications, 1:77-90 (1991); Blackwell et al, Science, 250: 1104-1110 (1990).
A wide variety of instrumentation has been developed for carrying out nucleic acid amplifications, particularly PCR, e.g. Johnson et al, U.S. Pat. No. 5,038,852 (computer controlled thermal cycler); Wittwer et al, Nucleic Acids Research, 17: 4353-4357 (1989)(capillary tube PCR); Hallsby, U.S. Pat. No. 5,187,084 (air-based temperature control); Garner et al, Biotechniques, 14: 112-115 (1993) (high-throughput PCR in 864-well plates); Wilding et al, International application No. PCT/US93/04039 (PCR in micro-machined structures); Schnipelsky et al, European Patent Application No. 90301061.9 (Publ. No. 0381501 A2)(disposable, single use PCR device), and the like. Important design goals fundamental to PCR instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-PCR processing steps, high speed cycling, minimization of sample volumes, real time measurement of amplification products, minimization of cross-contamination, or sample carryover, and the like.
In particular, the design of instruments that permit PCR to be carried out in closed reaction chambers and monitored in real time is highly desirable. Closed reaction chambers are desirable for preventing cross-contamination, e.g. Higuchi et al, Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al, PNAS(USA), 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives would severely reduce the value of the PCR-based procedure.
Real time monitoring of a PCR permits far more accurate quantitation of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the PCR. Real time monitoring also permits the efficiency of the PCR to be evaluated, which can indicate whether PCR inhibitors are present in a sample.
Holland, et al. and others have proposed fluorescence-based approaches to provide measurements of amplification products during a PCR. Holland et al, PNAS(USA), 88: 7276-7280 (1991). Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present (Higuchi et al, Biotechnology 10:413-417 (1992), Higuchi et al, Biotechnology 11:1026-1030 (1993), U.S. Pat. No. 5,210,015) or they have employed oligonucleotide probes that are cleaved during amplification by 5′ nuclease activity of the polymerase to release a fluorescent product whose concentration is a function of the amount of double stranded DNA present, commonly referred to as a 5′ nuclease assay. An example of a 5′ nuclease assay is the assay used in the Taqman™ LS-50 PCR Detection system (Perkin-Elmer).
In general, 5′ nuclease assays employ oligonucleotide probes labeled with at least one fluorescer and at least one quencher. Prior to cleavage of the probe, the at least one fluorescer excites the quencher(s) rather than producing a detectable fluorescence emission. The oligonucleotide probe hybridizes to a target oligonucleotide sequence for amplification in PCR or similar amplification reactions. The 5′→3′ nuclease activity of the polymerase used to catalyze the amplification of the target sequence serves to cleave the probe, thereby causing at least one fluorescer to be spatially separated from the one or more quenchers so that the signal from the fluorescer is no longer quenched. A change in fluorescence of the fluorescer and/or a change in fluorescence of the quencher due to the oligonucleotide probe being digested is used to indicate the amplification of the target oligonucleotide sequence.
In 5′ nuclease assays, it is often desirable to analyze a sample containing multiple different targets using a different spectrally resolvable species for each target. Such simultaneous detection of multiple targets in a single sample has a number of advantages over serial analysis of each of the targets. Because the sample is analyzed once, fewer steps are required for sample processing and only a single measurement is required. As a result, higher sample throughput and improved user convenience is achieved. In addition, by detecting multiple targets in a single sample, internal calibration is facilitated. An example of a process using simultaneous multispecies spectral detection is multicolor DNA sequencing where four spectrally resolvable fluorescent dyes are simultaneously detected.
One potential application for 5′ nuclease assays is in the area of screening for polymorphisms. Current diagnostic techniques for the detection of known nucleotide differences include: hybridization with allele-specific oligonucleotides (ASO) (Ikuta, et al., Nucleic Acids Research 15: 797-811 (1987); Nickerson, et al., PNAS (USA) 87: 8923-8927 (1990); Saiki, et al., PNAS (USA) 86: 6230-6234 (1989); Verlaan-de Vries, et al., Gene 50: 313-320 (1980); Wallace, et al., Nucleic Acids Research 9:879-894 (1981); Zhang, Nucleic Acids Research 19: 3929-3933 (1991)); allele-specific PCR (Gibbs, et al., Nucleic Acids Research 17: 2437-2448 (1989); Newton, et al., Nucleic Acids Research 17: 2503-2516 (1989)); solid-phase minisequencing (Syvanen, et al., American Journal of Human Genetics 1993; 52: 46-59 (1993)); oligonucleotide ligation assay (OLA) (Grossman, et al., Nucleic Acids Research 22: 4527-4534 (1994); Landegren, et al., Science 241: 1077-1080 (1988)); and allele-specific ligase chain reaction (LCR) (Abravaya, et al., Nucleic Acids Research 1995; 23: 675-682; Barany, et al., PNAS (USA) 88:189-193 (1991); Wu, et al., Genomics 4:560-569 (1989)). Genomic DNA is analyzed with these methods by the amplification of a specific DNA segment followed by detection analysis to determine which allele is present.
Lee, et al. has reported using PCR in combination with Taq polymerase to distinguish between different alleles at a single allelic site of the human cystic fibrosis gene. Lee, et al., Nucl. Acids Res. 21:3761-3766 (1993). Livak, et al. has reported distinguishing between alleles in the −23 A/T diallelic polymorphism of the human insulin gene where each allelic site was analyzed in a separate amplification reaction. Livak, et al., Nature Genetics, 9:341-342 (1995). Neither Lee, et al. nor Lival, et al. teach how to distinguish between alleles variants at two or more allelic sites in a single amplification reaction. A need currently exists for a method and instrumentation for distinguishing between multiple sets of substantially homologous sequences, such as allelic variants, in a single amplification reaction. The invention described herein provides such methods and instrumentation.